Method for isolating rat enteric nervous cells

Abstract

The application discloses a method for separating rat intestinal nerve cells, takes the intestinal muscle layer of a rat intestine and digests it into single cells, and then purifies the rat intestinal nerve cells by performing flow antibody staining and flow cytometry sorting on cell surface molecules such as CD9 and CD45, and comprises the following general steps: firstly, single cell preparation, secondly, single cell staining, and finally, flow cytometry separation. The application has a short time consumption and can realize real-time separation; the cell surface molecule markers for the rat intestinal nerve cells are determined to be CD45 negative and CD9 positive, and the rat intestinal nerve cells can be rapidly separated in real time for scientific research and clinical use.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a method for isolating rat intestinal nerve cells. Background Technology

[0002] The enteric nervous system is a complex system composed of nerve cells and glial cells in the intestinal wall. It autonomously regulates intestinal peristalsis, transmits nutrient information, senses dysbiosis, and works in concert with the intestinal immune system, gut microbiota, and other intestinal cells and tissues to maintain intestinal function and homeostasis. The enteric nervous system is associated with the development of various local or systemic diseases, including megacolon, achalasia, inflammatory bowel disease, cancer, diabetes, neurodevelopmental disorders, and neurodegenerative diseases.

[0003] Enteric nerve cells are a core component of the enteric nervous system. Establishing methods for isolating and purifying enteric nerve cells can be used in academic research to study their unique structure and function compared to central nervous system nerve cells; to explore their interactions with other types of intestinal cells; and to investigate their important role in the development and progression of related diseases. Clinically, they can be used to discover early biomarkers for related diseases; and to predict and assess patient responses to specific treatments and prognosis.

[0004] Existing nerve cell isolation techniques, including culture-based isolation, density gradient centrifugation, magnetic cell sorting, and microfluidics, are mainly used for the isolation of central nervous system nerve cells and are not suitable for the real-time isolation of intestinal nerve cells. This technology will effectively fill this gap. Summary of the Invention

[0005] The purpose of this invention is to provide a method for isolating rat intestinal nerve cells, which solves the problems existing in the prior art.

[0006] The technical solution adopted in this invention involves taking the intestinal muscular layer of rats and digesting it into single cells. Then, rat intestinal nerve cells are purified by flow cytometry staining and sorting of cell surface molecules such as CD9 and CD45. Existing nerve cell isolation technologies, such as cell culture separation, require several days and cannot guarantee real-time results; density gradient centrifugation is suitable for central nervous cells with large cell density differences but not for the intestine with relatively small cell density differences; magnetic cell sorting and microfluidic technology both require specific cell surface molecules as separation tags, but currently there are no molecular tags specifically for rat intestinal nerve cells.

[0007] Single-cell preparation:

[0008] 1. Euthanize an SD rat weighing 200-300g with carbon dioxide.

[0009] 2. Take rat large intestine, peel off the intermuscular nerve layer, transfer it to Librase enzyme at a concentration of 0.5-2 mg / ml, and digest in a water bath at 37°C for 20 minutes to 1 hour.

[0010] 3. Then transfer to trypsin and incubate in a 37°C water bath for 10–20 minutes.

[0011] 4. Centrifuge and discard the supernatant.

[0012] Flow cytometry sorting:

[0013] 1. Add antibody mixture containing three staining materials: antibody against cell surface tag CD9 (using a volume concentration of 1:200 to 1:1000), CD45 (1:200 to 1:1000), and the dead cell dye DAPI. Stain at 4°C or at room temperature in the dark for 20 minutes to 1 hour.

[0014] 2. Centrifuge, remove supernatant, add Neurobasal medium, and perform flow cytometry sorting. Cell populations that meet the criteria of being negative for the dead cell dye DAPI, negative for the cell surface molecular tag CD45, and positive for the cell surface molecular tag CD9 are considered high-purity intestinal nerve cells.

[0015] This invention offers a short processing time and enables real-time separation; it identifies CD45-negative and CD9-positive cell surface molecular tags for rat intestinal nerve cells. This invention allows for the rapid and real-time separation of rat intestinal nerve cells for research and clinical applications. Attached Figure Description

[0016] Figure 1 This is a flowchart of rat intestinal nerve cell isolation.

[0017] Figure 2 This is a diagram illustrating the in vitro culture of the isolated intestinal nerve cells. Detailed Implementation

[0018] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments.

[0019] Figure 1 The diagram shown is a flowchart of rat intestinal nerve cell isolation. The rat intestinal muscle layer was taken and digested into single cells. Then, the rat intestinal nerve cells were purified by flow cytometry antibody staining and flow cytometry sorting of cell surface molecules such as CD9 and CD45.

[0020] Figure 2 The image shows the in vitro culture of the isolated intestinal nerve cells: The isolated rat intestinal nerve cells were cultured in vitro. After adhesion, the cells were stained with the nerve cell tag Tuj1 and observed under a fluorescence microscope. It was confirmed that all the isolated cells were Tuj1 positive, that is, they were all intestinal nerve cells.

[0021] I. Single-cell preparation:

[0022] 1. Euthanize an SD rat weighing 200-300g with carbon dioxide.

[0023] 2. Take rat large intestine, peel off the intermuscular nerve layer, transfer it to Librase enzyme at a concentration of 0.5-2 mg / ml, and digest in a water bath at 37°C for 20 minutes to 1 hour.

[0024] 3. Then transfer to trypsin and incubate in a 37°C water bath for 10–20 minutes.

[0025] 4. Centrifuge and discard the supernatant.

[0026] II. Flow cytometry sorting:

[0027] 1. Add antibody mixture containing three staining materials: antibody against cell surface tag CD9 (using a volume concentration of 1:200 to 1:1000), CD45 (1:200 to 1:1000), and the dead cell dye DAPI. Stain at 4°C or at room temperature in the dark for 20 minutes to 1 hour.

[0028] 2. Centrifuge, remove supernatant, add Neurobasal medium, and perform flow cytometry sorting. Cell populations that meet the criteria of DAPI negative, CD45 negative, and CD9 positive are high-purity intestinal nerve cells.

[0029] Example 1:

[0030] Single-cell preparation:

[0031] 1. Euthanize a 200g SD rat with carbon dioxide.

[0032] 2. Take rat large intestine, peel off the intermuscular nerve layer, transfer it to Librase enzyme at a concentration of 1 mg / ml, and digest it in a water bath at 37°C for 40 minutes.

[0033] 3. Then transfer to trypsin and incubate in a 37°C water bath for 15 minutes.

[0034] 4. Centrifuge and discard the supernatant.

[0035] II. Flow cytometry sorting:

[0036] 1. Add antibody mixture containing antibody against cell surface tag CD9 (using a volume concentration of 1:400), CD45 (1:400), and the dead cell dye DAPI. Stain at 4°C in the dark for 40 minutes.

[0037] 2. Centrifuge, remove supernatant, add Neurobasal medium, and perform flow cytometry sorting, following the sorting gate method. Figure 1As shown, the selected cells are high-purity intestinal nerve cells.

[0038] Example 2:

[0039] Single-cell preparation:

[0040] 1. Euthanize a 250g SD rat with carbon dioxide.

[0041] 2. Take rat large intestine, peel off the intermuscular nerve layer, transfer it to Librase enzyme at a concentration of 0.5 mg / ml, and digest in a water bath at 37°C for 60 minutes.

[0042] 3. Then transfer to trypsin and incubate at 37°C for 10 minutes.

[0043] 4. Centrifuge and discard the supernatant.

[0044] Second-flow cytometer sorting:

[0045] 1. Add antibody mixture containing antibody against cell surface tag CD9 (using a volume concentration of 1:600), CD45 (1:800), and the dead cell dye DAPI. Stain at 4°C in the dark for 60 minutes.

[0046] 2. Centrifuge, remove supernatant, add Neurobasal medium, and perform flow cytometry sorting. Cell populations that meet the criteria of DAPI negative, CD45 negative, and CD9 positive are high-purity intestinal nerve cells.

[0047] Example 3:

[0048] Single-cell preparation:

[0049] 1. Euthanize a 300g SD rat with carbon dioxide.

[0050] 2. Take rat large intestine, peel off the intermuscular nerve layer, transfer it to Librase enzyme at a concentration of 1.5 mg / ml, and digest in a water bath at 37°C for 30 minutes.

[0051] 3. Then transfer to trypsin and incubate at 37°C for 20 minutes.

[0052] 4. Centrifuge and discard the supernatant.

[0053] Second-flow cytometer sorting:

[0054] 1. Add antibody mixture containing antibody against cell surface tag CD9 (using a volume concentration of 1:200), CD45 (1:1000), and the dead cell dye DAPI. Stain at room temperature in the dark for 20 minutes.

[0055] 2. Centrifuge, remove supernatant, add Neurobasal medium, and perform flow cytometry sorting. Cell populations that meet the criteria of DAPI negative, CD45 negative, and CD9 positive are high-purity intestinal nerve cells.

Claims

1. A method for isolating rat intestinal nerve cells, characterized in that, The myenteric nerve layer was removed from the large intestine of rats and digested into single cells. Then, the rat intestinal nerve cells were purified by flow cytometry antibody staining and flow cytometry sorting using cell surface molecules. The process includes the following steps: first, single cell preparation; second, single cell staining; and finally, flow cytometry separation. Single-cell preparation: 1) Take rat large intestine, peel off the intermuscular nerve layer, transfer it to Liberase enzyme at a concentration of 0.5-2 mg / ml, and digest it in a water bath for 20 minutes to 1 hour; 2) Transfer to trypsin and incubate at 37°C for 10–20 minutes; 3) Centrifuge at 1000-2000 rpm, discard the supernatant, and the resulting precipitate is a single cell; flow cytometry separation includes: performing flow cytometry sorting to sort cell populations that meet the criteria of being negative for the dead cell dye DAPI, negative for the cell surface molecular tag CD45, and positive for the cell surface molecular tag CD9.

2. The method for isolating rat intestinal nerve cells according to claim 1, characterized in that, Single-cell staining involves adding an antibody mixture to the resulting cell pellet and staining at 4°C or at room temperature in the dark for 20 minutes to 1 hour.

3. The method for isolating rat intestinal nerve cells according to claim 1 or 2, characterized in that, The stained cell pellet mixture was centrifuged at 1000–2000 rpm, the supernatant was removed, and the pellet was resuspended in Neurobasal medium for flow cytometry sorting.

4. The method for isolating rat intestinal nerve cells according to claim 1, characterized in that, SD rats weighing 200–300g.

5. The method for isolating rat intestinal nerve cells according to claim 2, characterized in that, Antibody mixtures refer to mixtures containing three dye components: antibodies that label the cell surface molecular tag CD9 with a fluorescent group; antibodies that label the cell surface molecular tag CD45 with a fluorescent group; and the dead cell dye DAPI.

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